Measurements of proteins are of central importance in biomedical research, and disease-related applications include early detection, causal identification, and treatment monitoring. Sensitive, specific, fast, inexpensive detection of multiple proteins in patient samples is critically important for future public health. The broad, long term goals of this project are to develop ultrasensitive new array devices for accurate measurements of panels of proteins. Recent research has shown that panels of 4 to 10 biomarker proteins, as opposed to the single biomarkers now used, will provide much more reliable cancer detection and monitoring. We will develop and optimize microfluidic arrays for proteins that are accurate, cheap, and can detect multiple proteins at levels 10-100 fold lower than any commercial alternatives. This project targets arrays for small panels of biomarker proteins in serum and tissue, not proteomic biomarker discovery or analysis. In the past funding period, we developed nanoparticle-based strategies for ultrasensitive protein immunoarrays using many thousands of enzyme labels on magnetic particles for electrochemical detection, or dye-nanoparticle labels for electro-optical detection. We utilized prototype sensors and arrays to detect up to four cancer biomarker proteins in the serum of prostate cancer patients with high accuracy. We recently combined nanostructured sensor arrays with microfluidics and off-line protein capture by the multilabel magnetic-antibody particles. This powerful approach greatly decreases interferences and can detect biomarker proteins in serum at levels of 100 fg mL-1 (~3 fM), 10-100 fold below capabilities of commercial assays. Such high sensitivity provides new opportunities for detection of biomarkers with inherently ultralow levels. This renewal project seeks to translate our ultrasensitive protein detection approaches into the realization of widespread clinical, surgical, and research applications. The deliverables are optimized, validated microfluidic array devices for ultrasensitive detection of virtually any small panel of proteins. A high-speed array for detection of cancer cell metastasis (spreading) to lymph nodes during cancer surgery will also be developed. For proof-of-concept, specific devices will be designed to predict the probability of oral cancer from collections of serum samples, and to clearly distinguish between viral (humanpapilloma virus) and non-viral oral cancers. Patient and control samples obtained by our NIDCR/NIH collaborators will be used to establish clinical sensitivity and selectivity of the immunoassay devices. Summarized specific aims are: (1) Optimize microfluidic systems featuring nanostructured sensor chips and multilabel amperometry for an 8-protein oral cancer panel; (2) Design and optimize a simple electro-optical microfluidic array using dye-nanoparticle labels detected by electrochemiluminescence (ECL); (3) Establish clinical sensitivity and selectivity for oral cancer diagnosis for the best array systems; (4) Develop a rapid microfluidic assay for detection of a metastasis biomarker in lymph nodes. 1